Dextran derivatives, preparation and medicinal applications

ABSTRACT

The invention concerns dextran derivatives, their applications as medicines with specific biological action, and their preparation method. Said derivatives correspond to the general formula DMC a B b SU c S d , in which: D represents a polysaccharide chain, preferably consisting of sequences of glucoside units, MC represents methylcarboxylate groups, B represents carboxymethylbenzylamide groups, Su represents sulphate groups, S represents sulphonate groups, a, b, c and d represent the degree of substitution (ds), respectively in groups, MC, B, Su and S; a being equal to 0 or ≧0.3, b being equal to 0 or ≧0.1, c being equal to 0 or ≧0.1 and d being equal to 0 or ≧0.1 provided that when d=0, a and/or b are not equal to 0, said products having homogeneity of chain size distribution, illustrated by a gauss elution profile symmetrical in high performance steric exclusion chromatography, and homogeneity in the distribution of charged chemical groups, illustrated by an elution profile with a single symmetrical peak in low pressure ion-exchange chromatography.

This application claims priority from PCT/FR98/02699 filed Dec. 11,1998, and from French patent application 97/15702 filed Dec. 11, 1997.

The present invention relates to dextran derivatives, and to theirapplications as medicines with specific biological action, such as acicatrizing action, an anti-complement action (substitute for plasma), aproliferation modulating action or an anticoagulant action, and morespecifically an anti-thrombotic action, as well as to a process fortheir preparation.

Various dextrans substituted with side chains bearing carboxylate andsulfonate groups have been described. In particular, French patent2,461,724 and French patent 2,555,589 describe dextrans substituted withsaid groups, which respectively display anticoagulant properties andanticoagulant and anti-inflammatory properties; European patent0,402,194 describes the cell and tissue regeneration properties of suchsubstituted dextrans.

It has also been shown that such substituted dextrans can have otherbiological activities, depending on the degree of substitution with saidgroups; in particular, European patent 0,514,449 describes dextrans (D)substituted with carboxymethyl (CM) and carboxymethylbenzylamidesulfonate (BS) groups of general formula D_(X)CM_(Y)BS_(Z), in which X,which represents the average number of unsubstituted saccharide unitsper 100 saccharide units, is less than or equal to 50, Y, whichrepresents the average number of carboxymethyl groups per 100 saccharideunits, is between 10 and 90, and Z, which represents the average numberof carboxymethylbenzylamide sulfonate groups per 100 saccharide units,is between 15 and 35; to give an agent for inhibiting the growth oftumor cells.

These various derivatives, the structure of which is summarized in FIG.1, are generally obtained by random substitution of dextran with threedifferent groups: carboxymethyl (CM), carboxymethylbenzylamide (B) andcarboxymethylbenzylamide sulfonate (S) (sulfonation on the aromatic ringwith chlorosulfonic acid).

More specifically:

a) the carboxymethylation of dextran (production of CMD) is carried outin basic aqueous medium, by the action of monochloroacetic acid. Threesuccessive carboxymethylation reactions are required to obtain a degreeof substitution (ds) of the dextran, expressed relative to the number offree hydroxyl functions in a glucoside unit of the dextran, of between0.7 and 1.1;

b) the coupling of benzylamine to the carboxymethyl groups (productionof CMDB) is based on the ability of the carboxylate function to form anunstable mixed anhydride capable of reacting with a reagent bearing aprimary amine function (R—NH₂). Two different processes or activationreactions were used to achieve the formation of a mixed anhydride:

action of isobutyl chloroformate (IBC) or

action of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

In both cases, the reaction is carried out in a heterogeneous medium:water/dimethylformamide or water/ethanol, respectively.

These two coupling processes give similar degrees of substitution (ds),of about 0.08 to 0.12, in a single step. In the case of coupling withEEDQ, the ds can reach 0.25 to 0.30, in a single step, when theintermediate product is activated at a temperature of 30 to 40° C.

As with the carboxymethylation, to achieve high ds values forbenzylamine, it is not possible to increase the concentrations of thevarious reagents. It is thus necessary to perform successive couplingsin order to improve the reaction yield. The CMDB precipitated, washedand dried after the first coupling undergoes a second and/or a thirdcoupling under exactly the same conditions as the first, withoutconsideration of the substitutions due to the first coupling.

c) the sulfonation or sulfation takes place by the action ofmonochlorosulfonic acid on the CMDB, in anhydrous organic medium (forexample dichloromethane) and in heterogeneous phase, the CMDB not beingsoluble in dichloromethane. In such a heterogeneous medium, thedistribution of the sulfates in the saccharide units occurs unequally(Ricketts, C. R., J. Chem. Soc., 1956, 3752-3756). It is necessary toperform the reaction in excess monochlorosulfonic acid, while at thesame time avoiding acid hydrolysis of the polysaccharide chain.

The [HSO₃Cl]/[bound B units] ratios for the CMDBs and [HSO₃Cl]/[free OH]ratios for the CMD range between 0.8 and 3.

In order essentially to obtain a sulfonation of the aromatic rings (Bunits), the [HSO₃Cl]/[bound B units] molar ratio should be equal to 3,whereas, in order to obtain a sulfation of the hydroxyl functions borneby the glucoside units (production of sulfate functions), the[HSO₃Cl]/[free OH] molar ratio is about 1. In any case, theconcentration of chlorosulfonic acid in the reaction medium should notexceed 0.15 M. Under these conditions, the percentage of units bearing asulfonate function depends on the percentage of units substituted withbenzylamide groups. A recent study (Maiga-Revel O. et al., CarbohydratesPolymers, 1997, 32, 89-93) has shown that the anticoagulant activity ofthe CMDSu (=carboxymethyldextran sulfate) and CMDBS(=carboxymethyldextran benzylamide sulfonate) dextran derivatives of theprior art depends on their sulfur content; however, CMDBSs have betteranticoagulant activity than that obtained with CMDSu's, for an identicalsulfur content.

The dextran derivatives obtained under the conditions defined above havethe drawback of having irregular distribution of the chemical groups andof the sizes of the polysaccharide chains, leading to a heterogeneousfinal product whose properties are difficult to control.

The inventors have developed a novel process for preparing dextranderivatives, which allows better control of the production ofspecifically defined products (controllable degree of substitution,homogeneity of the distribution of the charged or uncharged chemicalgroups and size homogeneity of the polysaccharide chains in the finalproduct, selection and better reproducibility of the desired activity).

A subject of the present invention is dextran derivatives of generalformula DMC_(a)B_(b)Su_(c)S_(d), in which:

D represents a polysaccharide chain, preferably consisting ofconcatenations of glucoside units,

MC represents methylcarboxylate groups,

B represents carboxymethylbenzylamide groups,

Su represents sulfate groups (sulfation of the-free hydroxyl functionsborne by the glucoside units),

S represents sulfonate groups (sulfonation of the aromatic rings),

a, b, c and d represent the degree of substitution (ds), expressedrelative to the number of free hydroxyl functions in a dextran glucosideunit, with groups MC, B, Su and S, respectively; a being equal to 0 or≧0.3, b being equal to 0 or ≧0.1, c being equal to 0 or ≧0.1 and d beingequal to 0 or ≧0.15, with the proviso that when d=0, a and/or b are ≠0,

which products display:

homogeneity of the size distribution of the chains, illustrated by anelution profile of symmetrical Gaussian type in high performance stericexclusion chromatography, and

homogeneity of the distribution of charged chemical groups, illustratedby an elution profile as a single symmetrical peak in low-pressure ionexchange chromatography.

These products are considered as being copolymers consisting of fictivesubunits R—OH and R—OX, it being possible for X to be amethylcarboxylate (MC) benzylamide (B), sulfate (Su) or sulfonate (S)group, the polysaccharide chain of the unsubstituted dextran beingconsidered as consisting of 300 fictive R—OH subunits, instead of 100glucoside units, with regard to the fact that an unsubstituted glucosideunit comprises three free hydroxyl groups. Thus, a dextranmethylcarboxylate (DMC) with a degree of substitution (ds) withmethylcarboxylate groups of 1.2 contains 1.20 substituted groups (R—MC)and 1.80 free hydroxyl groups (R—OH), per glucoside unit.

This thus gives, in contrast with the heterogeneous products of theprior art, homogeneous products, of targeted composition, in which thebioactive chemical groups are distributed along the macro-molecularchains in a specific order, giving the product a biological propertywhich will not be found in a product of the same overall composition butin which the distribution of said groups along the macromolecular chainsis different (different preparation, in particular).

In other words, in the dextran derivatives according to the invention,the distribution of the chemical groups gives the final product aspecific biological property; the consequence of such a distribution isthat the chemical composition of each polysaccharide chain is identicalto the overall chemical composition of the product. Accordingly, thereis an optimum chemical composition for a maximum specific biologicalactivity; there is thus a direct relationship between the biologicalproperty considered and the overall chemical composition of the product.

For example:

the dextran derivatives of general formula DMC_(a)B_(b)Su_(c)S_(d) asdefined above, in which a≧0.6, b≠0, c equal to 0 or ≦0.5 and d≦0.15 or dequal to 0 are essentially cicatrizing agents, preferably when they havea molar mass between 3000 and 500,000 g/mol; the dextran derivativespreferred as cicatrizing agents are those in which a is between 0.7 and0.9; c≦0.5 and d≦0.15 or equal to 0;

the dextran derivatives of general formula DMC_(a)B_(b)Su_(c)S_(d) asdefined above, in which a≧0.3, b≠0, c equal to 0 or ≦0.4 and d≦0.15 orequal to 0 are essentially agents with anti-complement activity andplasma substitutes, preferably when they have a molar mass of between10,000 and 60,000 g/mol; the dextran derivatives preferred as agentswith anti-complement activity and as substitutes for plasma are those inwhich a is between 0.40 and 1.15, b≦0.4, c≦0.2 and d≦0.15 or equal to 0;

the dextran derivatives of general formula DMC_(a)B_(b)Su_(c)S_(d) asdefined above, in which a≧0.5, b≠0, c equal to 0 or ≦0.4 and d≦0.15 areessentially agents for modifying cell proliferation, preferably whenthey have a molar mass of between 3000 and 100,000 g/mol; the dextranderivatives preferred as cell proliferation modulating agents are thosein which a is between 0.5 and 1.2; b is between 0.2 and 0.6; c isbetween 0.1 and 0.4 and d≦0.15 or equal to 0; and

the dextran derivatives of general formula DMC_(a)B_(b)Su_(c)S_(d) asdefined above, in which a≧0.4, c≧0.3 and d≦0.15 or equal to 0 areessentially anticoagulant agents, preferably when they have a molar massof between 3000 and 20,000 g/mol and a value for b≠0.

A subject of the present invention is also medicines, characterized inthat they comprise as active principle at least one dextran derivativeas defined above, optionally combined with another active principleand/or with at least one pharmaceutically acceptable vehicle and/or aphysiologically acceptable support, preferably a liposome.

The combined active principles are chosen from the group comprisingdextrans, growth factors (for example an acidic fibroblast growth factor(FGF) or a basic FGF), local anesthetics, anti-infection agents, sericproteins and collagen.

A subject of the present invention is also a process (process 1) forpreparing dextran derivatives of general formulaDMC_(a)B_(b)Su_(c)S_(d), as defined above, characterized in that itcomprises the following steps:

a) carboxymethylation comprising (i) activation of an unsubstituteddextran, by placing said dextran in contact with a basic two-phaseliquid aqueous-alcoholic medium for at least 1 h with stirring, (ii)addition of monochloroacetic acid to the activated product obtained, ata temperature of between 40 and 90° C., preferably at 60° C., the ratioR_(MC), equal to the number of moles of monochloroacetic acid/number ofmoles of OH, being between 0.3 and 2, (iii) isolation and optionallypurification of the dextran methylcarboxylate (DMC) obtained.

b) coupling of benzylamine with methylcarboxylate groups(benzylamidation) comprising (i) the placing in contact, for at least 2h and in an acidic aqueous medium, of the DMC obtained in a) with aprimary amine (benzylamine), in the presence of a water-solublecarbodiimide such as 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeta-p-toluene sulfonate (CMC) or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) ascoupling agent, at a temperature of between 0° C. and 30° C., the CMC/MCmolar ratio being between 0.25 and 2 and the benzylamine/MC molar ratiobeing between 0.25 and 2, (ii) isolation of the dextran methylcarboxylbenzylamide (DMCB) obtained and optionally purification thereof.

Such a step, performed in homogeneous medium and in the presence of awater-soluble carbodiimide as coupling reagent, allows better control ofthe reaction and thus preparation of the final product, this producthaving a homogeneity of the chain size distribution, illustrated by anelution profile of symmetrical Gaussian type in high performance stericexclusion chromatography and a homogeneity of the distribution ofcharged chemical groups, illustrated by an elution profile as a singlesymmetrical peak in low-pressure ion exchange chromatography.

c) sulfation comprising (i) the formation of a trialkylammonium salt ofthe DMCB obtained in b), (ii) solubilization of the salt obtained in ananhydrous polar solvent, generally a Lewis base (electron donor), suchas dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and (iii)addition, to said dissolved salt, of a complex based on sulfur trioxidesuch as SO₃-pyridine, SO₃-triethylamine or SO₃-DMF dissolved in the samesolvent, at a temperature of less than 70° C., the complex based onsulfur trioxide/free OH molar ratio being between 0.25 and 12, andoptionally

d) sulfonation of the groups B by mixing, with stirring, a derivativeDMCBSu in suspension in an anhydrous solvent with chlorosulfonic aciddissolved in the same solvent, at a temperature between room temperatureand the boiling point of the solvent used.

Unexpectedly, the process according to the invention allows the degreeof substitution of the dextran to be controlled, in a number of stepswhich is significantly smaller than the number of steps in the processesof the prior art, to give products with an elution profile ofsymmetrical Gaussian type in high performance steric exclusionchromatography and an elution profile as a single symmetrical peak inlow-pressure ion-exchange chromatography and the desired biologicalactivity, with yields which are significantly higher than those of theprior art.

In addition, the process according to the invention makes it possible toreproducibly synthesize a product of desired chemical composition forthe biological property selected. Thus, a biological activity preferablycorresponds to a given chemical composition of a product.

For example, step a) gives, in a single step, a ds with MC of 1.0±0.1per glucoside unit and a yield of greater than or equal to 80%, for anR_(MC) value=0.85, in a water/tert-butanol or water/isopropanol (15/85v/v) mixed medium, with stirring for 2 hours at 60° C.; starting with aDMC with a ds with MC=1, for a CMC/MC molar ratio of 0.75 and abenzylamine/MC molar ratio of 1, and with stirring at room temperaturefor 16 hours, step b) gives a DMCB product which has a ds with MC of0.70±0.05 and a ds with B of 0.30±0.03, with a yield of greater than80%; and step c) gives a yield of greater than or equal to 60%.

According to one advantageous embodiment of said process, in step a),the water/alcohol ratio is between 10/90 (v/v) and 25/75 (v/v) and ispreferably 15/85 (v/v).

As a variant, the process (process 2) for preparing the dextranderivatives of general formula DMC_(a)B_(b)Su_(c)S_(d), as definedabove, in which a=0 or a≠0, b is other than 0 and d=0, comprises thefollowing steps:

a) preparation of N-methylphenyl-2-chloroacetamide by placing abenzylamine in contact with chloroacetyl chloride, followed by isolationand purification of the product,

b) placing dextran, dissolved in basic aqueous-alcoholic solution, incontact successively with N-methylphenyl-2-chloroacetamide in alcoholicsolution obtained in a) in the presence or absence of monochloroaceticacid in alcoholic solution, maintaining the mixture obtained at atemperature above 40° C., preferably at 60° C., followed by isolationand optionally purification of the DB or DMCB obtained, and

c) sulfation of the product obtained in b) comprising (i) formation of atrialkylammonium salt, (ii) dissolution of the salt obtained in ananhydrous polar solvent, generally a Lewis base, such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF), (iii) addition, to saiddissolved salt, of a complex based on sulfur trioxide such asSO₃-pyridine, SO₃-triethylamine or SO₃-DMF dissolved in the samesolvent, at a temperature below 70° C., the complex based on sulfurtrioxide/free OH molar ratio being between 0.25 and 12, and (iv)isolation and optionally purification of the DBSu or DMCBSu obtained.

The presence of steps b) and c) above makes it possible to obtain thesame homogeneity of the final product as that obtained with process 1above.

In order to prepare the dextran derivatives of general formulaDMC_(a)B_(b)Su_(c)S_(d), as defined above, in which a is other than 0,b=0 and d=0 (→DMCSu) (process 3), said process is characterized in thatit comprises the following steps:

a) carboxymethylation of an unsubstituted dextran, under the conditionsoutlined above, and

b) sulfation of the DMC obtained in a) under the conditions outlinedabove.

Besides the preceding arrangements, the invention also comprises otherarrangements, which will emerge from the description which follows, withreference to examples for carrying out the process which is the subjectof the present invention, as well as to the attached drawings, in which:

FIG. 1 schematically illustrates the structure of a dextran substitutedwith various chemical groups attached to the glucoside units; theposition of the substituent on the various carbons of the glucoside baseunits is shown in 2, by way of example;

FIG. 2 illustrates the high performance steric exclusion chromatogramobtained with a dextran derivative as defined above (DMCBSu);

FIG. 3 illustrates the ion-exchange chromatogram obtained with a dextranderivative as defined above (DMCBSu).

However, it should be clearly understood that these examples are givenpurely for the purposes of illustration of the subject of the invention,of which they do not in any way constitute a limitation.

EXAMPLE 1 Physicochemical Characterizations of the Products Obtained

Acid-base Assay

The acidimetric assay gives an estimation of the number ofmilliequivalents of carboxymethyl groups and establishes the compositionof the final products.

Procedure

A titrating calibration determines the molarity of the sodium hydroxideusing a standard: potassium monohydrogen phthalate.

Between 15 and 25 mg of purified, dried (under vacuum at 40° C.) andvery accurately weighed product are dissolved in a water/acetone (50/50v/v) mixture so as to obtain a solution with a final concentration ofabout 0.25 mg/ml.

The apparent pH, which is then between 7 and 8, is lowered to between2.8 and 3, using 10% HNO₃ solution. The titrating solution is added bymeans of a precision automatic microburette with a total volume of 5 ml.At least 3 assays are carried out on each product and the results,expressed as milliequivalents of carboxymethyl functions per gram ofpolymer, make it possible to calculate the degree of substitution withcarboxymethyl groups. This method also makes it possible to estimate thedegree of substitution with units B, by means of the difference in thenumber of milliequivalents of carboxymethyl functions before and afterbenzylamidification. For all of the products, the compositions wereestablished with the aid of elemental analysis.

Nitrogen, Sulfur and Chlorine Elemental Analysis

Assaying the nitrogen and sulfur by elemental analysis, expressed asgrams of element per 100 g of product, makes it possible to determine,respectively, the percentage of substitution with benzylamide (B),sulfonate (S) and sulfate (Su) groups.

Assaying the chlorine makes it possible to check the degree of purity ofthe samples, the percentage of salt (NaCl) not being allowed to exceed 1to 2% after the purification.

Desulfation of the Products (DCMBSuS)

The product in the form of sodium salt (250 mg, 10 ml) is stirred slowlyat room temperature with 3 ml of cation-exchange resin (Amberlite IR120H⁺, 16-45 mesh, total exchange capacity: 1.9 meq/ml). After 2 h, theacidic solution is filtered, neutralized with pyridine (1 to 2 ml) to apH of 6-6.5 and evaporated to dryness. The pyridinium salt obtained istaken up 3 times with 10 ml of anhydrous methanol and evaporated todryness.

The residue is dispersed in 25 ml of a 90/9/1 mixture of dimethylsulfoxide (DMSO), methanol and pyridine. The solution is stirred in anoil bath heated at 90° C. for 72 h. The reaction is stopped by adding 20ml of cold double-distilled water and the mixture is then neutralizedwith aqueous 1M NaOH solution. The desulfated product is purified by lowpressure steric exclusion chromatography on a Sephadex G15 column andthen diafiltered on a cell equipped with a membrane with a cutoffthreshold of 1000 Da. Between 160 and 210 mg of desulfated product areobtained.

Determination of the Molar Mass

The molar mass of the products prepared is determined by highperformance steric exclusion chromatography (HPSEC). Three columnsystems were used:

two columns mounted in series (Si300 diol and Hema Sec Bio 40) coveringa fractionation range between 106 and 5000 g/mol,

a Zorbax GF450 column (exclusion range: 450,000-20,000 g/mol),

a TSK gel G2000 column (exclusion range: 40,000-1000 g/mol).

They are calibrated using a kit of pullulans (853,000 to 5800 g/mol);enzymatic dextran (oligosaccharide, 1500 g/mol); melezitose(trisaccharide, 522 g/mol); sucrose (disaccharide, 342 g/mol) andglucose (monosaccharide, 180 g/mol). The samples to be analyzed areprepared (at 2 mg/mL) and eluted with a solution of 0.15 M NaCl, 0.05 MNa₂HPO₄ buffered to pH 7.3. After filtration through a filter ofporosity 0.2 μm, 100 μl of the sample are injected, the flow rate beingadjusted by means of a Waters model 510 pump and detection at the columnoutlet being performed by differential refractometry. The chromatogramsprocessed by the GPC Chromstar analysis software make it possible todetermine the chromatographic molar mass (Mc), the weight-average molarmass ({overscore (Mp)}) and the number-average molar mass ({overscore(Mn)}) as well as the polydispersity index of the products.

Fourier Transform Infrared Spectroscopy (FTIR)

1 mg of product to be analyzed is mixed with a solution of 150 mg of KBrin 1 ml of double-distilled water. The mixture is filtered through afilter of porosity 0.45 μm, frozen and lyophilized; a disk is preparedwith the powder obtained and the spectrum is recorded immediately. Theapparatus is an FTIR spectrophotometer (Perkin Elmer model 1600) and thespectra are processed on computer with the IRDM software (Perkin Elmer).

EXAMPLE 2 Preparation of Various DMCBSu's With b= or b≠0

Table I below shows the various derivatives obtained and theirproperties, established after analysis of the final product.

TABLE I Composition Major Degree of Molar bio- Synthesis substitution ±10% mass logical Product conditions MC B Su (g/mol) property DMCBSu1 a0.75 0.20 0.15 48,000 SP DMCBSu2 b 0.80 0.25 0.15 70,000 SP DMCBSu3 c1.10 0.40 0.40 59,000 C DMCBSu4 d 0.70 0.30 0.25 48,000 SP DMCBSu5 e0.80 0.35 0.15 56,000 PM DMCBSu6 f 0.60 0.50 0.30 63,000 PM DMCBSu7 g0.70 0.30 0.16 70,000 PM DMCBSu8 h 0.70 0.20 0.35 67,000 SP DMCSu i 1.000 0.37 70,000 AC SP = substitute for plasma, C = cicatrizing agent, AC =anticoagulant, PM = cell proliferation modulator.

a) DMCBSu1

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 isdispersed in 612 ml of isopropanol in a jacketed 2 l reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml of double-distilledwater in a 500 ml beaker. The solution obtained is cooled to 4° C. andadded slowly to the dextran-isopropanol mixture. This mixture is stirredfor 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 1 h 30 min.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.289 mol) of DMC with a degree of substitution with MC of 1.0±0.1are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. The mixture is cooledto 4° C. 62.3 g of coupling agent (CMC) are added rapidly thereto(CMC/CM molar ratio=0.5). Once the coupling agent is fully dissolved inthe mixture, 16 ml of benzylamine are added rapidly (benzylamine/CMmolar ratio=0.5). The mixture is then stirred for 16 hours at roomtemperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this concentrated solution are frozen and lyophilizedfor the analyses. The degrees of substitution with MC and B are,respectively, 0.79±0.07 and 0.22±0.03.

Sulfation

Sulfation requires the preparation of the triethylammonium salt of DMCB.

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.0.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.39 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 24.5 g (0.154 mol) (complex/free OHmolar ratio=0.4) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the solution of triethylammonium salt of DMCB. Themixture is stirred for 2 h at room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and ultrafiltered to constant volume withdouble-distilled water on a membrane with a cutoff threshold of 5000g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu1 are thus obtained. The elemental analysisof the sulfur gives a ds with sulfate groups of 0.15±0.02.

b) DMCBSu2

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system. 67.9 g (1.7 mol)of NaOH are dissolved in 188 ml in a 500 ml beaker. The solutionobtained is cooled to 4° C. and added slowly to the dextran-isopropanolmixture. This mixture is stirred for 1 hour.

87.5 g (0.93 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio =3) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.28 mol) of DMC with a degree of substitution with MC of 1.1±0.1are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. The mixture is cooledto 4° C. 85.7 g of coupling agent (CMC) are added rapidly thereto(CMC/CM molar ratio=0.7). Once the coupling agent is fully dissolved inthe mixture, 22.2 ml of benzylamine are added rapidly (benzylamine/CMmolar ratio=0.7). The mixture is then stirred for 16 hours at roomtemperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this concentrated solution are frozen and lyophilizedfor the analyses. The degrees of substitution with MC and B are,respectively, 0.85±0.07 and 0.27±0.03.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.0.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.365 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 22.9 g (0.144 mol) (complex/free OHmolar ratio=0.4) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu2 with a degree of substitution with Sugroups of 0.15±0.02 are thus obtained.

c) DMCBSu3

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and purified:

either by means of a double precipitation in 3 l of methanol. Theprecipitate thus recovered is washed twice with methanol and dried in anoven under vacuum at 40° C.,

or by tangential filtration through a membrane with a cutoff thresholdof 5000 daltons.

The purified solution is then concentrated and lyophilized. 70 g (0.289mol) of DMC with a degree of substitution with MC of 1.0±0.1 areobtained.

Recarboxymethylation of the DMC

The 70 g (0.289 mol or 0.578 mol of free OH) of DMC obtained aredispersed in 1035 ml of isopropanol in a 3 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

46.4 g (1.16 mol) of NaOH are dissolved in 265 ml in a 500 ml beaker.The solution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

54.6 g (0.578 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/free OH molar ratio=1) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 30 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses. 75 g (0.26 mol) ofDMC with a degree of substitution with MC of 1.58±0.12 are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. 169 g of couplingagent (CMC) are added rapidly thereto (CMC/CM molar ratio=1). Once thecoupling agent is fully dissolved in the mixture, 43.7 ml of benzylamineare added rapidly (benzylamine/CM molar ratio=1). The mixture is thenstirred for 16 hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 22 g/l(3 l). 20 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,1.15±0.10 and 0.44±0.04.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH of close to 7.

The neutralized solution is concentrated and lyophilized. About 70 g oftriethylammonium salt are obtained.

The 70 g (0.24 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 30.7 g (0.193 mol) (complex/free OHmolar ratio=0.8) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 65 g of DMCBSu3 with a degree of substitution with sulfategroups of 0.40±0.04 are thus obtained.

d) DMCBSu4

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.284 mol) of DMC with a degree of substitution with MC of1.05±0.10 are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. 126.5 g of couplingagent (CMC) are added rapidly thereto (CMC/CM molar ratio=0.8). Once thecoupling agent is fully dissolved in the mixture, 32.6 ml of benzylamineare added rapidly (benzylamine/CM molar ratio=0.8). The mixture is thenstirred for 16 hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,0.74±0.06 and 0.33±0.03.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.0.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.38 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 30.2 g (0.154 mol) (complex/free OHmolar ratio=0.5) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu4 with a degree of substitution with Sugroups of 0.25±0.03 are thus obtained.

e) DMCBSu5

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 5 l hour.

117.2 g (1.24 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=4) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.271 mol) of DMC with a degree of substitution with MC of1.2±0.11 are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. 124.1 g of couplingagent (CMC) are added rapidly thereto (CMC/CM molar ratio=0.9). Once thecoupling agent is fully dissolved in the mixture, 35.5 ml of benzylamineare added rapidly (benzylamine/CM molar ratio=0.9). The mixture is thenstirred for 16 hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to 35 pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,0.84±0.07 and 0.38±0.04.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.

The neutralized solution is concentrated and lyophilized. About 67 g oftriethylammonium salt are obtained.

The 67 g (0.33 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 21 g (0.132 mol) (complex/free OHmolar ratio=0.4) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 62 g of DMCBSu5 with a degree of substitution with sulfategroups of 0.15±0.02 are thus obtained.

f) DMCBSu6

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

87.5 g (0.93 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio 3) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.28 mol) of DMC with a degree of substitution with MC of 1.1±0.10are obtained.

1st benzylamidation

The pH of the above solution is adjusted to 4.75. 130.5 g of couplingagent (CMC) are added rapidly thereto (CMC/CM molar ratio=1). Once thecoupling agent is fully dissolved in the mixture, 30.3 ml of benzylamineare added rapidly (benzylamine/CM molar ratio=0.9). The mixture is thenstirred for 16 hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The solution is concentrated to a volume of2.1 l (˜28 g/l or 0.1 mol/l). 20 ml of this solution are frozen andlyophilized for the analyses. The degrees of substitution with MC and Bare, respectively, 0.75±0.06 and 0.38±0.04.

2nd benzylamidation

The pH of the 2.1 l of the above DMCB solution is readjusted to 4.75.The mixture is cooled to 4° C. 40 g of coupling agent (CMC) are addedrapidly thereto (CMC/CM molar ratio=0.5). Once the coupling agent isfully dissolved in the mixture, 10.3 ml of benzylamine are added rapidly(benzylamine/CM molar ratio=0.5). The mixture is then stirred for 16hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,0.65±0.06 and 0.54±0.05.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.365 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 34.9 g (0.219 mol) (complex/free OHmolar ratio=0.6) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu6 with a degree of substitution with sulfategroups of 0.30±0.03 are thus obtained.

g) DMCBSu7

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.284 mol) of DMC with a degree of substitution with MC of 1.05are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. 126.5 g of couplingagent (CMC) are added rapidly thereto (CMC/CM molar ratio=0.8). Once thecoupling agent is fully dissolved in the mixture, 32.6 ml of benzylamineare added rapidly (benzylamine/CM molar ratio=0.8). The mixture is thenstirred for 16 hours at room temperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 20 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,0.74±0.06 and 0.33±0.03.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.38 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 24.2 g (0.154 mol) (complex/free OHmolar ratio=0.4) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu7 with a degree of substitution with sulfategroups of 0.16±0.02 are thus obtained.

h) DMCBSu8

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system. 67.9 g (1.7 mol)of NaOH are dissolved in 188 ml in a 500 ml beaker. The solutionobtained is cooled to 4° C. and added slowly to the dextran-isopropanolmixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.295 mol) of DMC with a degree of substitution with MC of0.94±0.10 are obtained.

Benzylamidation

The pH of the above solution is adjusted to 4.75. The mixture is cooledto 4° C. 58.8 g of coupling agent (CMC) are added rapidly thereto(CMC/CM molar ratio=0.5). Once the coupling agent is fully dissolved inthe mixture, 15.2 ml of benzylamine are added rapidly (benzylamine/CMmolar ratio=0.5). The mixture is then stirred for 16 hours at roomtemperature.

The mixture is then ultrafiltered to constant volume on a membrane witha cutoff threshold of 5000 g/mol, successively with 15 l of osmosedwater to pH 7, then with 10 l of 0.05 M, pH 9.6 carbonate buffer andfinally with double-distilled water until the conductivity of thefiltrate is satisfactory. The concentration of this solution is 21 g/l(3 l). 5 ml of this solution are frozen and lyophilized for theanalyses. The degrees of substitution with MC and B are, respectively,0.75±0.07 and 0.22±0.03.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.

The neutralized solution is concentrated and lyophilized. About 65 g oftriethylammonium salt are obtained.

The 65 g (0.4 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 45.2 g (0.154 mol) (complex/free OHmolar ratio=0.7) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 60 g of DMCBSu8 with a degree of substitution with sulfategroups of 0.35±0.04 are thus obtained.

i) DMCSu

Carboxymethylation

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor maintainedat room temperature and fitted with a stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol (acid/OH molar ratio=2.5) in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 h.

The aqueous phase is then recovered and diluted until a volume of 2.1 lis obtained. The solution thus obtained is purified to constant volumeon a membrane with a cutoff threshold of 5000 g/mol, withdouble-distilled water, until the conductivity of the filtrate issatisfactory. The concentration of the solution is 28 g/l. 20 ml of thissolution are frozen and lyophilized for the analyses.

70 g (0.289 mol) of DMC with a degree of substitution with MC of 1.0±0.1are obtained.

Sulfation

The solution obtained above is eluted through a column of IR 120 H⁺cation-exchange resin (1.5 l). The solution thus acidified isneutralized with triethylamine to a pH close to 7.

The neutralized solution is concentrated and lyophilized. About 80 g oftriethylammonium salt are obtained.

The 80 g (0.466 mol of free OH) of salt obtained are dried in an ovenunder vacuum at 40° C. for 5 h, along with any required materials. Thissalt is then dissolved, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system, in 1.7 l of DMSOpredried over 4 Å molecular sieves. 52 g (0.326 mol) (complex/free OHmolar ratio=0.7) of SO₃-pyridine complex are dissolved in 300 ml of DMSOand added slowly to the polymer solution. The mixture is stirred for 2 hat room temperature under 5 argon.

The reaction is stopped by adding 2 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then diluted until a DMSO concentrationof 0.5% by volume is obtained, and is ultrafiltered to constant volumewith double-distilled water on a membrane with a cutoff threshold of5000 g/mol. The solution is then concentrated to a volume of 5 l andultrafiltered to constant volume on the same membrane, successively with10 l of 0.05 M, pH 9.6 carbonate buffer and with double-distilled wateruntil the conductivity of the filtrate is satisfactory. The solutionthus purified is concentrated to one-seventh of its volume, frozen andlyophilized. 70 g of DMCSu are thus obtained. Elemental analysis of thesulfur gives a ds with sulfate groups of 0.37±0.04.

EXAMPLE 3 Preparation of a DMCBSuS

A suspension of 10 9 of DMCBSu, obtained in accordance with Example 2,in 350 ml of anhydrous dichloromethane is prepared. 0.62 ml ofchlorosulfonic acid is added to 35 ml of anhydrous dichloromethane(chlorosulfonic acid/units B molar ratio=1). The mixture is stirredunder argon for 1 hour at room temperature. The product is filteredthrough a No. 4 sinter funnel and washed successively with 200 ml ofdichloromethane, 200 ml of 50/50 (v/v) dichloromethane/dioxane andfinally with pure dioxane.

The DMCBSuS in acid form is immediately dissolved in 150 ml of water andthe pH of the solution is adjusted to 9 with 6 M NaOH and maintained atthis value for 1 hour. The solution is then neutralized with 0.1 M HC1,frozen and lyophilized.

The amount of sulfonates is established by the difference between thesulfur contents of the initial and final products after desulfation ofthe two products, under the conditions specified in Example 1.

EXAMPLE 4 Process Variant for the Preparation of a DMCBSu.

a) Preparation of the Reagent N-methylphenyl-2-chloroacetamide

109.4 ml (1 mol) of benzylamine are mixed with 1 l of ether predriedover 4 Å molecular sieves, in a three-necked round-bottomed flask fittedwith a stirring system and an argon circulation system (inertatmosphere).

79.7 ml (1 mol) of chloroacetyl chloride are added dropwise(introduction time of 30 minutes), using a dropping funnel. After theaddition, the flask is sealed and stirred at room temperature for 1hour.

The mixture is then washed successively with 1.5 l of 0.5 M citric acidsolution (220 g), 1.5 l of double-distilled water at 4° C., 1.5 l of 0.5M sodium hydrogen carbonate solution (63 g) and 1.5 l of 1 M NaClsolution (87.8 g).

The ether phase is recovered and evaporated to dryness.

The dry residue is purified by double-recrystallization from water:

the residue is dissolved in double-distilled water (1-2 w/v% solution)brought to the boiling point. This boiling solution is filtered using aNo. 4 sinter funnel preheated to 100° C. The filtered solution is cooledand stored at 4° C. overnight. The reagent is then recovered on Wattmanpaper and dried in an oven under vacuum at about 50° C.

b) Synthesis of DMCB

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 400 ml of isopropanol in a 2 l jacketed reactor fitted witha stirring system.

56.5 g (1.41 mol) of NaOH are dissolved in 225 ml in a 500 ml beaker.The solution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. The mixture is stirred for 1 hour. 84.9 g(0.46 mol) of the N-methylphenyl-2-chloroacetamide obtained in a) aredissolved in

675 ml of isopropanol in a 1 l beaker. 43.7 g (0.46 mol) ofmonochloroacetic acid are dissolved in 200 ml of isopropanol in another,500 ml beaker.

The N-methylphenyl-2-chloroacetamide solution is introduced rapidly intothe jacketed reactor and the mixture is brought to 60° C. with the aidof a thermostat connected to the reactor. About 5 minutes later, themonochloroacetic acid solution is rapidly added thereto and the mixtureis stirred at 60° C. for 2 hours.

The aqueous phase is then recovered and purified:

either by means of a double precipitation in 3 l of methanol; theprecipitate thus recovered is washed twice with methanol and dried in anoven under vacuum at 40° C.

or by tangential filtration through a membrane with a cutoff thresholdof 5000 daltons; the purified solution is then concentrated andlyophilized.

The composition of the DMC obtained by this method is as follows:MC=0.90±0.09 and B=0.30±0.03.

c) Synthesis of DMCBSu

Formation of the Tributylammonium (or Triethylammonium) Salt

50 g (0.18 mol) of the DMCB obtained in step b) are dissolved in 2 l ofdouble-distilled water. The solution obtained is eluted through a columnof cation-exchange resin (Amberlite IR 120 H⁺). The product thusacidified is neutralized with 10% tributylamine in ethanol (ortriethylamine) to a pH close to 7.

The neutralized solution is purified by tangential filtration through amembrane with a cutoff threshold of 5000 daltons, concentrated andlyophilized. About 60 g of tributylammonium (or triethylammonium) saltare obtained.

Sulfation of the Tributylammonium (or Trithylammonium) DMCB

The 60 g of salt obtained are dried in an oven under vacuum at 40° C.for 4 hours, along with any materials required. The salt is thendissolved in a three-necked round-bottomed flask fitted with a stirringsystem and an argon circulation system, in 2.7 l ofN,N-dimethylformamide (DMF) predried over 4 Å molecular sieves (5.7 lfor the triethylammonium salt). 38.7 g (0.24 mol) of SO₃-pyridinecomplex (46.6 g (0.29 mol) in the case of the triethylammonium salt) aredissolved in 300 ml of DMF and added slowly to the polymer solution. Themixture is stirred for 2 hours at room temperature.

The reaction is stopped by adding 3 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then ultrafiltered through a membranewith a cutoff threshold of 5000 daltons, concentrated and lyophilized.

EXAMPLE 5 Synthesis of DMCSu

a) Synthesis of DMC

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 612 ml of isopropanol in a 2 l jacketed reactor fitted witha stirring system.

67.9 g (1.7 mol) of NaOH are dissolved in 188 ml in a 500 ml beaker. Thesolution obtained is cooled to 4° C. and added slowly to thedextran-isopropanol mixture. This mixture is stirred for 1 hour.

72.9 g (0.77 mol) of monochloroacetic acid are dissolved in 450 ml ofisopropanol in a 1 l beaker.

The monochloroacetic acid solution is introduced rapidly into thejacketed reactor and the mixture is brought to 60° C. with the aid of athermostat connected to the reactor. The mixture is maintained at thistemperature, with stirring, for 2 hours.

The aqueous phase is then recovered and purified:

either by means of a double precipitation in 3 l of methanol; theprecipitate thus recovered is washed twice with methanol and dried in anoven under vacuum at 40° C.,

or by tangential filtration through a membrane with a cutoff thresholdof 5000 daltons; the purified solution is then concentrated andlyophilized.

b) Synthesis of DMCSu

Formation of the Tributylammonium (or Triethylammonium) Salt

50 g (0.18 mol) of DMC are dissolved in 2 l of double-distilled water.The solution obtained is eluted through a column of cation-exchangeresin (Amberlite IR 120 H⁺). The product thus acidified is neutralizedwith 10% tributylammonium [sic] in ethanol (or triethylamine) to a pHclose to 7.

The neutralized solution is purified by tangential filtration through amembrane with a cutoff threshold of 5000 daltons, concentrated andlyophilized. About 60 g of tributylammonium (or triethylammonium) saltare obtained.

Sulfation of Tributylammonium (or Triethylammonium) DMC

The 60 g of salt obtained are dried in an oven under vacuum at 40° C.for 4 hours, along with any required materials. The salt is thendissolved, in a three-necked round-bottomed flask fitted with a stirringsystem and an argon circulation system, in 2.7 l ofN,N-dimethylformamide (DMF) predried over 4 Å molecular sieves. 44.8 g(0.28 mol) of SO₃-pyridine complex (or 55.8 g (0.35 mol) in the case ofthe triethylammonium salt) are dissolved in 300 ml of DMF and addedslowly to the polymer solution. The mixture is stirred for 2 hours atroom temperature under argon.

The reaction is stopped by adding 3 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then ultrafiltered through a membranewith a cutoff threshold of 5000 daltons, concentrated and lyophilized.

The composition of a DMCSu obtained is as follows: MC=1±0.1,Su=0.37±0.04.

EXAMPLE 6 Synthesis of a DBSu

a) Synthesis of DB

50 g (0.31 mol) of dextran T40 with a molar mass of about 40,000 aredispersed in 400 ml of isopropanol in a 2 l jacketed reactor fitted witha stirring system.

56.5 g (1.41 mol) of NaOH are dissolved in 225 ml in a 500 ml beaker.The solution obtained is cooled to 4° C. and added slowly to thedextranisopropanol mixture. This mixture is stirred for 1 hour.

84.9 g (0.46 mol) of N-methylphenyl-2-chloroacetamide (see Example 5)are dissolved in 875 ml of isopropanol in a 1 l beaker. The solutionobtained is introduced rapidly into the jacketed reactor and the mixtureis brought to 60° C. with the aid of a thermostat connected to thereactor; the mixture is maintained at this temperature, with stirring,for 2 hours.

The aqueous phase is then recovered and purified:

either by means of a double precipitation in 3 l of methanol; theprecipitate thus recovered is washed twice with methanol and dried in anoven under vacuum at 40° C.;

or by tangential filtration through a membrane with a cutoff thresholdof 5000 daltons; the purified solution is then concentrated andlyophilized.

About 60 g of DB are obtained. The composition of the product is asfollows: B=0.3±0.003.

b) Synthesis of DBSu

The 60 g of DB obtained are dried in an oven under vacuum at 40° C. for4 hours, along with any materials required. The product is thendispersed, in a three-necked round-bottomed flask fitted with a stirringsystem and an argon circulation system, in 2.7 l ofN,N-dimethylformamide (DMF) predried over 4 Å molecular sieves. 40.7 g(0.26 mol) of SO₃-pyridine complex are dissolved in 300 ml of DMF andadded slowly to the polymer solution. The mixture is stirred for 2 hoursat room temperature under argon.

The reaction is stopped by adding 3 l of double-distilled water at 4° C.to the mixture and the pH of the medium is brought to 7.5-8 using 2 MNaOH solution. The solution is then ultrafiltered through a membranewith a cutoff threshold of 5000 daltons, concentrated and lyophilized.

The composition of a DBSu obtained is as follows: B=0.3±0.03 andSu=0.14±0.02.

EXAMPLE 7 Anticoagulant and Anti-complement Activity of the ProductsAccording to the Invention

a) Measurement of the Anticoagulant Activity

The activated cephalin time is an exploratory test which is sensitive toall the factors of the endogenous pathway (thrombin, V, VIII, IX, XI andXII) of the coagulation system except for platelet factor III. Thereagent used (APTT, Organon Teknika) contains rabbit brain phospholipids(platelet factor III) and a particulate activator (micronized silica orelagic acid). The procedure used is as follows:

100 μl of platelet-poor human plasma (PPP)

100 μl of Owren Koller buffer (OKB)

100 μl of APTT reagent

incubation for 3 min at 37° C.

100 μl of 25 mM calcium chloride (CaCl₂)

measurement of the time for appearance of the clot.

A curve is plotted, of the logarithm of the coagulation time as afunction of the polymer concentration. The slope of the curve makes itpossible to calculate the specific anticoagulant activity of thepolysaccharide according to the equation:${a\quad ( {{IU}\text{/}{mg}} )} = {\frac{{slope}\quad {of}\quad {the}\quad {DMCBSu}\quad {curve}}{{slope}\quad {of}\quad {the}\quad {heparin}\quad {curve}} \times \begin{matrix}{{specific}\quad {anticoagulant}\quad {activity}} \\{{of}\quad {the}\quad {heparin}\quad {standard}\quad {used}}\end{matrix}}$

b) Measurement of the Inhibition of the Activation of Complement

The action of dextran derivatives of variable chemical composition wasstudied in a hemolytic or CH50 assay, according to a procedure in whichthe human serum is incubated with one of the dextran derivatives of theinvention for 30 minutes at 37° C. before being activated with sheeperythrocytes sensitized with rabbit anti(sheep red blood cells)antibodies (EA). By definition, one CH50 unit corresponds to theconcentration of complement proteins (contained in one milliliter ofserum) capable of inducing the hemolysis of 50% of 2×10⁷ activated EAsin a reaction medium in which the volume, the temperature and thereaction time are kept constant. The number of hemolytic sites per cellis calculated.

The activity in terms of the inhibition of complement activation isexpressed as a percentage of inhibition of the formation of convertaserelative to the control tube in which the number of hemolytic sites isdetermined in the absence of dextran derivative.

This activity, A, is expressed as weight of product (at constant volume)(μg), required for 50% inhibition of the formation of hemolytic sites.This expression of the anti-complement activity means that it isinversely proportional to the weight of product. In other words, theanti-complement activity is proportionately higher the lower the weightof product.

This activity varies as a function of the overall chemical compositionof the product.

The results obtained with 5 derivatives according to the invention areillustrated in Table II below, which recalls their overall chemicalcomposition and their specific anticoagulant activity, a, expressed ininternational units per mg of product (relative to heparin at 173IU/mg).

TABLE II Anti- Anti- complement coagulent Chemical activity activitySample composition A a reference MC B Su μg IU/mg Dextran 0 0 0 1250  0Dextran MC 1.0 0 0 570 0 DMCBSu1 0.75 0.20 0.15 150 0.02 DMCBSu2 0.800.25 0.15 150 0.04 DMCBSu3 1.10 0.40 0.40  35 4.0 DMCBSu4 0.70 0.30 0.25125 0.08 DMCB1 0.80 0.10 0 300 0 Heparin — — — 2700  173

It is found that the anti-complement activity increases as the ds withMC and Su units increases. Above a ds with MC units of about 0.40, theanticoagulant activity increases as the ds with Su units increases.

It is observed that the derivatives with low values of ds with Su unitsalready have high anti-complement activity, whereas their anticoagulantactivity is very low. Finally, it is noted that the presence of MC and Bunits alone induces slight anti-complement activity, whereas it has noanticoagulant effect.

These results show that it is possible to obtain products endowed withhigh inhibitory activity on the activation of complement and very lowspecific anticoagulant activity.

EXAMPLE 8 Antiproliferative Activity of the Compounds According to theInvention

The DMCBSu compounds have stimulatory or inhibitory effects on cellgrowth, depending on the nature of the cells. Thus, for example in thecardiovascular field, the products have the advantage of stimulating thegrowth of endothelial cells and inhibiting the growth of smooth musclecells (SMC). These properties are important in pharmacology, their majoradvantage being the prevention of restenosis after angioplasty orcardiovascular surgical interventions.

The antiproliferative activity of DMCBSu's was studied on smooth musclecells (SMC) of rat aorta. 24 hours after inoculating the cells in amedium containing 10% fetal calf serum (FCS), the cells are subjected todeficient conditions for 3 days (medium containing 0.1% FCS) (thisallows the cells to become synchronized by stopping them at the GO/G1stage of the cell cycle). The cells are then fed again with mediumcontaining 10% FCS, also containing variable concentrations of the testproduct (from 0 to 1 mg/ml). A control is prepared under the sameconditions but without the addition of product.

After growing them for 5 days, the cells are counted (using an automaticcounter or by measuring the radioactivity in counts per minute afterincorporating tritiated thymidine) and the inhibition is determined asfollows, the abbreviation “nb” denoting the word “number”:

%I=(1−(nb of Cells in the Presence of Product)/(nb of Cells WithoutProduct))×100

The anticoagulant activity of the various products was also determined.

Table III below collates the results obtained for two DMCBSu's as wellas for native dextran and heparin.

TABLE III Anticoagulant Composition (ds) activity I Product MC B SuIU/mg (%) Dextran 0 0 0 0  0 DMCBSu6 0.60 0.50 0.30 5.0 80 DMCBSu5 0.800.35 0.15 3.0 85 Heparin 173 80

These results show that, for DMCBSu6 and DMCBSu5, the inhibition (I) ofthe cell growth of smooth muscle cells is comparable to, or even greaterthan, that of heparin. However, these two products have markedly loweranticoagulant activity than that of heparin.

EXAMPLE 9 Potentiation of Cell Growth in the Presence of FibroblastGrowth Factors (FGF)

The proliferative activity of the DMCBSu's was evaluated on 2 celllines:

CCL39: Chinese hamster lung fibroblast line.

HUCVEC: endothelial cells of the human umbilical cord vein.

The growth tests are carried out by maintaining the cells underdeficient conditions, i.e. in a culture medium containing 0% or 2% fetalcalf serum, followed by incorporating the test product at differentconcentrations.

The countings are carried out using an automatic counter or by measuringthe radioactivity, in counts per minute, after incorporating tritiatedthymidine.

The capacity of the products to potentiate and protect certain growthfactors such as FGFs is evaluated according to the following procedure.

The mitogenic activity of the FGFs studied is measured by means of abiological activity test (dose-response test) in order to determine theED₅₀ (amount of growth factor required to obtain half the maximumincorporation of tritiated thymidine into the cells).

This ED₅₀ is 5 ng/ml for the CCL39 cells and 2 ng/ml for the HUCVECcells.

These ED₅₀ values are used for the rest of the studies.

For the HUCVEC cells, the combination of FGF (2 ng/ml) with 400 μg/ml ofDMCBSu5 has the same mitogenic power as the FGF alone at 20 ng/ml.

For the CCL39 cells, in the absence of DMCBSu, the ED₅₀ is about 5 ng/mlof FGF. In the presence of 1000 μg/ml of DMCBSu1, the ED₅₀ falls to 0.8ng/ml.

These results show the potentiating effect of the cell growth factorsconsidered by the DMCBSu's. This effect is linked to the overallchemical composition of the products. The table below gives, forexample, the overall chemical composition of particularly activeDMCBSu's.

COMPOSITIONS OF DMCBSU's (in ds) MC B Su DMCBSu1 0.75 0.20 0.15 DMCBSu50.80 0.35 0.15 DMCBSu3 1.10 0.40 0.40

As emerges from the preceding account, the invention is not in any waylimited to its implementation, preparation or application methods whichhave just been described in greater detail; on the contrary, itencompasses all the variants which may occur to a person skilled in theart, without departing from the context or scope of the presentinvention.

What is claimed is:
 1. Dextran derivatives of general formulaDMC_(a)B_(b)Su_(c)S_(d), in which: D represents a polysaccharide chain,comprising concatenations of glucoside units, MC representedmethylcarboxylate groups, B represents carboxymethylbenzylamide groups,Su represents sulfate groups, S represents sulfonate groups, a, b, c andd represent the degree of substitution (ds), expressed relative to thenumber of free hydroxyl functions in a dextran glucoside unit, withgroups MC, B, Su and S, respectively; a being equal to 0 or ≧0.3, bbeing equal to 0 or ≧0.1, c being equal to 0 or ≦0.1 and d being equalto 0 or ≦0.15, with the proviso that when d=0, a and/or b are ≠0, whichproducts display: homogeneity of the size distribution of the chains,illustrated by an elution profile of symmetrical Gaussian type in highperformance steric exclusion chromatography, and homogeneity of thedistribution of charged chemical groups, illustrated by an elutionprofile as a single symmetrical peak in low pressure ion exchangechromatography.
 2. Dextran derivatives of general formulaDMC_(a)B_(b)Su_(c)S_(d), according to claim 1, characterized in thata≧0.6, b≠0, c equal to 0 or ≦0.5 and d≦0.05 or equal to 0 and in thattheir molar mass is between 3000 and 500,000 g/mol.
 3. Dextranderivatives of general formula DMC_(a)B_(b)Su_(c)S_(d), according toclaim 1, characterized in that a≧0.3, b≠0, c equal to 0 or ≦0.4 andd≦0.15 or equal to 0 and in that their molar mass is between 10,000 and60,000 g/mol.
 4. Dextran derivatives of general formulaDMC_(a)B_(b)Su_(c)S_(d), according to claim 1, characterized in thata≧0.5, b≠0, c equal to 0 or ≦0.4 and d≦0.15 or equal to 0 and in thattheir molar mass is between 3000 and 100,000 g/mol.
 5. Dextranderivatives of general formula DMC_(a)B_(b)Su_(c)S_(d), according toclaim 1, characterized in that a≧0.4, b≠0, c≧0.3 and d≦0.15 or equal to0 and in that their molar mass is between 3000 and 20,000 g/mol.
 6. Amedicine comprising, as an active principle, at least one dextranderivative according to claim
 1. 7. Process for preparing dextranderivatives of general formula DMC_(a)B_(b)Su_(c)S_(d), according toclaim 1, comprising the following steps: a) carboxymethylationcomprising (i) activation of an unsubstituted dextran, by placing saiddextran in contact with a basic two-phase liquid aqueous-alcoholicmedium for at least 1 hour with stirring, (ii) addition ofmonochloroacetic acid to the activated product obtained, at atemperature of between 40 and 90° C., preferably at 60° C., the ratioRMC, equal to the number of moles of monochloroacetic acid/number ofmoles of OH, being between 0.3 and 2, (iii) isolation and optionallypurification of the dextran methylcarboxylate (DMC) obtained; b)coupling of benzylamine with methylcarboxylate groups comprising (i) theplacing in contact, for at least 2 hour and in an acidic aqueous medium,of the DMC obtained in a) with benzylamine, in the presence of awater-soluble carbodiimide as coupling agent, at a temperature ofbetween 0° C. and 30° C., the CMC/MC molar ratio being between 0.25 and2 and the benzylamine/MC molar ratio being between 0.25 and 2, (ii)isolation of the dextran methylcarboxyl benzylamide (DMCB) obtained andoptionally purification thereof, c) sulfation comprising (i) theformation of a trialkylammonium salt of the DMCB obtained in b), (ii)solubilization of the salt obtained in an anhydrous polar solvent,generally a Lewis base, and (iii) addition, to said dissolved salt, of acomplex based on sulfur trioxide such as SO₃-pyridine, SO₃-triethylamineor SO₃-DMF dissolved in the same solvent, at a temperature of less than70° C., the complex based on sulfur trioxide/free OH molar ratio beingbetween 0.25 and 12; and, optionally, d) sulfonation of the groups B bymixing, with stirring, a derivative DMCBSu in suspension in an anhydroussolvent with chlorosulfonic acid dissolved in the same solvent, at atemperature between room temperature and the boiling point of thesolvent used.
 8. Process according to claim 7, characterized in that, instep a), the water alcohol ratio in said two phase liquidaqueous-alcoholic medium is between 10/90 v/v and 25/75 v/v.
 9. Processaccording to claim 7 characterized in that the water-solublecarbodiimide from step b) is selected from the group consisting of1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meta-p-toluene sulfonate(CMC) and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride(EDC).
 10. Methylcarboxydextran benzylamide (DB).
 11. A medicine asclaimed in claim 6 further comprising at least one additional activeprincipal selected from the group consisting of an acidic fibroblastgrowth factor (FGF) and a basic fibroblast growth factor (FGF).
 12. Amedicine as claimed in claim 6 further comprising at least one member ofthe group consisting of at least one pharmaceutically acceptable vehicleand a physiologically acceptable support.
 13. A process as claimed inclaim 7 wherein said Lewis base is at least one member selected from thegroup consisting of dimethyl sulfoxide (DMSO) and dimethylformamide(DMF).
 14. A process as claimed in claim 8 wherein said water alcoholratio in said two phase liquid aqueous-alcoholic medium is about 15/85v/v.